Immunoassays have found widespread application in the field of clinical diagnostics for the detection and measurement of drugs, vitamins, hormones, proteins, metabolites, microorganisms, and other substances of interest (analytes) in biological and non-biological fluids. Typically, these analytes occur in micromolar (10.sup.-6 M) or less concentration.
Immunoassays generally incorporate antibodies and antigens as reactants, at least one of which is labeled with a signal-producing compound (e.g., radioisotope, fluorophore, enzyme, etc.). Following mixture with the sample and incubation, specific antibody/antigen reactions occur (specific binding). The reaction mixture is subsequently analyzed to detect free and specifically bound labeled reactant, enabling a measurement of the analyte in the sample.
Immunoassays can be divided into two general categories, homogeneous and heterogeneous. In a homogeneous immunoassay, the signal emitted by the specifically bound labeled reactant is different from the signal emitted by the free labeled reactant. Hence bound and free can be distinguished without physical separation.
The archetypal homogeneous immunoassay is the enzyme-multiplied immunoassay technique (EMIT) which is disclosed in U.S. Pat. No. 3,817,837. In this technology, analyte present in patient sample and analyte/enzyme conjugate compete for a limited amount of anti-analyte antibody. Specific binding of antibody to the conjugate modulates its enzymatic activity, hence the amount of enzyme activity is proportional to the amount of analyte in the sample.
Homogeneous immunoassays have the advantages of being rapid, easy to perform, and readily amenable to automation. Their principal disadvantages are that they are relatively prone to interferences, are generally limited to low molecular weight analytes, and are generally limited in sensitivity to approximately 10.sup.-9 M.
In a heterogeneous immunoassay, the signal emitted by the bound labeled reactant is indistinguishable from the signal emitted by the free labeled reactant; therefore, a separation step is required to distinguish between the two. Typical heterogeneous immunoassays include the radioimmunoassay (RIA) and the enzyme-linked immunosorbent assay (ELISA).
In the RIA, radiolabeled analyte and analyte present in patient sample compete for a limited amount of immobilized (solid-phase) anti-analyte antibody. The solid phase is washed to remove unbound, labeled analyte, and either the bound or the free fraction is analyzed for the presence of labeled reactant. ELISA assays are performed analogously. In the latter case though, the signal is an enzyme instead of a radioisotope. Heterogeneous immunoassays typically employ at least one reactant immobilized on a solid phase. Solids used to immobilize reactants in immunoassays have included controlled pore glass and preformed polymers, such as polyvinyls, polyacrylamides, polydextrans, and polystyrenes. Numerous separation methods are known in the art and have been used in heterogeneous immunoassays. These include centrifugation, microfiltration, affinity chromatography, and gel-permeation chromatography. Since the kinetics of reaction between an immobilized antibody (or antigen) and its binding site tend to be slower than the kinetics of the same reaction occurring in solution, long incubation times are frequently required. When the multiple wash steps often needed are considered, it can be appreciated that heterogeneous assays tend to be time-consuming and labor-intensive. However, they are in general more sensitive than homogeneous assays and less prone to interferences, since interfering substances can be removed in the wash step(s).
More recently, EP No. 124,050 by Jolley, discloses a method of solid phase immunoassay in which the analyte is reacted with an immunoreactant immobilized on water-insoluble particles in a substantially suspended state and thereafter concentrated by mirofiltration to a volume substantially less than the volume of the original sample. This method suffers from the same disadvantages associated with all solid phase-based immunoassays, namely, slow reaction kinetics and high nonspecific binding.
In addition to the immunoassays described above, there have more recently been a number of efforts directed toward the use of reversibly soluble "complexes" within the context of immunoassays. Representative of the earlier work with reversibly-soluble enzyme catalysts is the research conducted by Charles, et al., Biotech., Bioeng., 16: 1553 (1974), which discloses the synthesis of reversibly soluble lysozyme-alginic acid "complexes" by first reacting alginic acid with CNBr and then contacting the activated acid with a solution of lysozyme. The resultant complex is soluble above pH 4.0 and can be rendered insoluble by lowering the pH below 3.0. Because the pH optimum of lysozyme is 8.5, however, the pH change required to effect insolubilization is quite large (pH being on a logarithmic scale). Such a large change, with concomitant changes in ionic strength, would be unlikely to be well tolerated by the majority of biologically active materials. Furthermore, most specific binding pairs will dissociate at a pH of 3; thus the method as disclosed is not widely applicable for catalysis and is not applicable at all to specific binding assays.
An improvement over the complexes of Charles, et al., is the work of Margolin et al. (Biotech., Bioeng., 24: 237 (1982)), which discloses the synthesis of reversibly soluble enzyme-polyelectrolyte complexes by first reacting an enzyme with an activated polycation and then reacting the enzyme-polycation conjugate with a polyanion. Solubility of the resultant complexes is a function of pH or salt concentration. These complexes overcome some of the disadvantages of Charles et al., supra, in tht the precipitation occurs over a narrower and less extreme range of pH or salt concentration. Nonetheless, the method remains cumbersome due to the need to complex oppositely charged polyelectrolytes and is expected to be prone to nonspecific binding via ionic interactions with the complex.
One of the first direct applications of the use of reversibly-soluble complexes to immunoassays is disclosed in U.S. Pat. No. 4,088,538, issued to schneider. The Schneider patent discloses a process for using and preparing a reversibly soluble, enzymatically active enzyme product which consists of an enzyme covalently bonded to a water-soluble organic polymer selected from polyacrylic acid, dextran, carboxymethyl cellulose, and polyethylene glycol, which have carboxyl or amino side groups that impart to the complex its reversible solubility. Insolubilization is effected by a change in pH or calcium ion concentration. However, the suitability of this method to the immunoassay of substances in biological fluids is uncertain.
Another application similar to that disclosed within Schnieder is U.S. Pat. No. 4,530,900, issued to Marshall et al. Marshall et al. discloses the use of reversibly soluble polymers of alginic acid to which antibodies or antigens have been covalently attached in an enzyme immunoassay. Polymers which find utility in this method are those which have free carboxyl groups and which can be precipitated from solution by a change in pH or by the addition of certain metal ions, such as calcium. The enzymatic signal is developed in solution after separation of bound from free labeled component and redissolution of the precipitate.
There is a need in the art, however, for an immunoassay method which is sensitive to sub-micromolar concentrations of analyte, which has fast-reaction kinetics, which permits multiple analyses to be performed on a single sample, and which is readily amenable to automation. The present invention fulfills this need, and further provides other related advantages.